Methods for alleviating deleterious effects of 3-deoxyglucosone

ABSTRACT

Disclosed is a class of compounds which inhibit the enzymatic conversion of fructose-lysine into fructose-lysine-3-phosphate in an ATP dependent reaction in a recently discovered metabolic pathway. According to the normal functioning of this pathway, fructose-lysine-3-phosphate (FL3P) is broken down to form free lysine, inorganic phosphate and 3-deoxyglucosone (3DG), the latter being a reactive protein modifying agent. 3DG can be detoxified by reduction to 3-deoxyfructose (3DF), or it can react with endogenous proteins to form advanced glycation end-product modified proteins (AGE-proteins). Also disclosed are therapeutic methods of using such inhibitors to alleviate deleterious effects of 3-deoxyglucosone (3DG).

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 09/974,323, filed Oct. 10, 2001 which is a continuation-in-partof U.S. patent application Ser. No. 09/182,114, filed Oct. 28, 1998,which is a continuation-in-part of U.S. application Ser. No. 09/095,953,filed Jun. 11, 1998, now abandoned, which is a continuation-in-part ofInternational Application No. PCT/US98/02192, filed Feb. 5, 1998, whichis a continuation-in-part of U.S. application Ser. No. 08/794,433, filedFeb. 5, 1997, now U.S. Pat. No. 6,004,958. The entire disclosure of eachof the aforesaid patent applications is incorporated by referenceherein.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY-SPONSOREDRESEARCH AND DEVELOPMENT

Pursuant to 35 U.S.C. §202(c), it is hereby acknowledged that the U.S.Government has certain rights in the invention described herein, whichwas made-in part with funds from the National Institutes of Health(Grant Nos. DK44050, DK50317, DK50364, and DK55079)

BACKGROUND OF THE INVENTION

The present invention relates to therapeutic agents and their use forthe treatment of diabetes, and in particular for preventing, reducing ordelaying the onset of diabetic complications and other disorders ofrelated etiology, such as glycogen storage diseases, including Fanconi'ssyndrome. More particularly, the present invention relates to a class ofenzyme inhibitors which inhibit the enzymatic conversion of fructoselysine (FL) to fructose-lysine-3-phosphate (FL3P), which is believed tobe an important step in the biochemical mechanism leading to diabeticcomplications. This invention also relates to a method of assessing adiabetic patient's risk of experiencing diabetic complications, as wellas a method of determining the efficacy of therapeutic intervention inpreventing, reducing or delaying the onset of diabetic complications.

There are four particularly serious complications of diabetes, namely,diabetic nephropathy or kidney disease; diabetic retinopathy whichcauses blindness due to destruction of the retina; diabetic neuropathyinvolving the loss of peripheral nerve function; and circulatoryproblems due to capillary damage. Both retinopathy and nephropathy arethought to be subsets of the general circulatory problems associatedwith this disease state. The role of microvascular dysfunction in latestage diabetes has been recently summarized (Tooke, Diabetes, 44: 721(1995)). Throughout this disclosure, the terms “diabetes-associatedpathologic conditions” and synonymous terms are meant to include thevarious well-known retinopathic, neuropathic, nephropathic,macroangiopathic, as well as other complications of diabetes anddiseases of related etiology, including glycogen storage diseases.

The similarities between the pathologies arising from diabetes and thoseresulting from aging have been extensively reported. Studies have shownthat many diabetes-associated pathologic conditions are clinically verysimilar to the pathologies normally associated with aging. It has beenshown, for example, that in diabetes arteries and joints prematurelystiffen, lung elasticity and vital capacity prematurely decrease.Moreover, atherosclerosis, myocardial infarction and strokes occur morefrequently in diabetics than in age-matched non-diabetic individuals.Diabetics are also more susceptible to infections, and are more likelyto have hypertension, accelerated bone loss, osteoarthritis and impairedT-cell function at a younger age than non-diabetics.

The similarities between diabetes-associated 15′ pathologic conditionsand aging would appear to suggest a common mechanistic rationale. Avariety of mechanisms have been proposed as a common biochemical basisfor both diabetes-associated pathologic conditions and aging. Thehypothesis most strongly supported by data from human subjects ispremised on a non-enzymatic glycosylation mechanism. This hypothesisstates that the aging process and diabetes-associated pathologicconditions, such as those described above, are caused, at least in part,by protein modification and cross-linking by glucose and glucose-derivedmetabolites via the Maillard reaction (Monnier et al., Proc. Natl. Acad.Sci. USA, 81: 583 (1984) and Lee et al., Biochem. Biophys. Res. Comm.,123: 888 (1984)). The modified proteins resulting from suchglycosylation reactions are referred to herein as advanced glycation endproduct-modified proteins (AGE-proteins). It is widely accepted that3-deoxyglucosone (3DG) is a key intermediate in the multi-step reactionsequence leading to AGE-protein formation. 3DG is a glucose-derivedmetabolite that can react with proteins leading to the cross-linking ofboth intracellular and extracellular proteins, such as collagen andbasement membranes.

In the case of diabetic complications, the reactions that lead toAGE-proteins are thought to be kinetically accelerated by the chronichyperglycemia associated with this disease. Evidence supporting thismechanism includes data showing that long-lived proteins such ascollagen and lens crystallins from diabetic subjects contain asignificantly greater AGE-protein content than do those from age-matchednormal controls. Thus, the unusual incidence of cataracts in diabeticsat a relatively early age is explainable by the increased rate ofmodification and cross-linking of lens crystalline. Similarly, the earlyonset of joint and arterial stiffening, as well as loss of lung capacityobserved in diabetics is explained by the increased rate of modificationand cross-linking of collagen, the key structural protein. Because theseproteins are long-lived, the consequences of modification tend to becumulative.

Another factor demonstrating cause and effect relationship betweendiabetic complications and hyperglycemia is hyperglycemic memory. Oneparticularly striking example of this phenomenon is the development ofsevere retinopathy in dogs that were initially diabetic, then treated torestore normal blood glucose levels. Although the dog eyes werehistologically normal at the time of the treatment, over time diabeticretinopathy developed in these animals in spite of the normalizedglucose concentrations (Engerman et al., Diabetes, 36: 808 (1987)).Thus, the underlying damage to the eyes irreversibly occurred during theperiod of early hyperglycemia, before clinical symptoms were evident.

Diabetic humans and animals have been shown to have higher than normalconcentrations of early and late sugar modified AGE-proteins. In fact,the increase in AGE-proteins is greater than the increase in bloodglucose levels. The concentration of AGE-proteins can be estimated byfluorescence measurement, as some percentage of sugar moleculesrearrange to produce protein-bound fluorescent molecules.

The pathogenic role of AGE-proteins is not limited to diabetes. Proteinglycation has been implicated in Alzheimer's disease (Harrington et al.,Nature, 370: 247 (1994)). Increased protein fluorescence is also seenwith aging. Indeed, some theories trace the aging process to acombination of oxidative damage and sugar-induced protein modification.Thus, a therapy that reduces AGE-protein formation may also be useful intreating other etiologically-similar human disease states, and perhapsslow the aging process.

It has generally been assumed that the formation of AGE-proteins beginswith the reaction of a protein amino group and a sugar, primarilyglucose. One typical literature citation states “The initial adductformed by glycation of ε-amino groups of lysine residues is the Amadoricompound, fructoselysine. Glycation is an initial step in a complexseries of reactions, known collectively as the Maillard or browningreaction, which ultimately leads to the formation of crosslinked,precipitated, oxidized, brown and fluorescent proteins”. K. J. Knecht etal., Archives of Biochem. Biophys., 294: 130 (1992).

The formation of AGE-proteins from sugars is a multi-step process,involving early, reversible reactions with sugars to producefructose-lysine containing proteins. These modified proteins thencontinue to react to produce irreversibly modified AGE-proteins. It isclear that AGE-proteins are not identical to proteins containingglycated-lysine residues, as antibodies raised against AGE-proteins donot react with fructose-lysine. It is also clear that AGE-proteins existas multiple chemical species; however few have been identified. Thechemical species ε-Amino-(carboxymethyl)lysine has been identified asone important final AGE-protein structure in recent studies (Reddy etal., Biochem., 34: 10872 (1995) and Ikeda et al., Biochemistry, 35: 8075(1996)). This study failed to chemically identify another AGE-proteinepitope that made up approximately 50% of the modified sites. A methodof studying the kinetics of AGE-protein formation from ribose hasrecently been developed (Khalifah et al., Biochemistry, 35: 4645(1996)). However, this study suggests that ribose may play an importantphysiological role in AGE-protein formation, supporting the relativelybroad definitions of glycated-lysines and fructose-lysine providedbelow.

Other references point out the distinction between proteins containingglycated lysine residues and AGE proteins, “Equilibrium levels ofSchiff-base and Amadori products are reached in hours and weeks,respectively. The reversible, equilibrium nature of early glycosylationproducts is important, because it means that the total amount of suchproducts, even on very long-lived proteins, reaches a steady-stateplateau within a short period of time. Since these early glycosylationproducts do not continue to accumulate on collagen and other stabletissue proteins over years in chronic diabetes, it is not surprisingthat their concentration does not correlate with either the presence orthe severity of diabetic retinopathy. . . . Some of the earlyglycosylation products on collagen and other long-lived proteins of thevessel walls do not dissociate, however. Instead, they undergo a slow,complex series of chemical rearrangements to form irreversible advancedglycosylation end products”. M. Brownlee et al., New England Journal ofMedicine, 318: 1315 (1988). The only route for production of thesemodified proteins which is described in the scientific literatureinvolves an initial reaction between proteins and sugar molecules.

Numerous references point out that the formation of AGE-proteins occursthrough a multi-step pathway and that 3-deoxyglucosone (3-DG) is a keyintermediate in this pathway. M. Brownlee, Diabetes, 43: 836 (1994); M.Brownlee, Diabetes Care, 15: 1835 (1992); T. Niwa et al., Nephron, 69:438 (1995); W. L. Dills, Jr., Am. J. Clin. Nutr., 58: S779 (1993); H.Yamadat et al., J. Biol. Chem., 269: 20275 (1994); N. Igaki et al.,Clin. Chem., 36: 631 (1990). The generally accepted pathway forformation of 3DG from the reaction of sugars and proteins is illustratedin FIG. 1. As can be seen in FIG. 1, a sugar (glucose) moleculeinitially forms a Schiff base with a protein-lysine amino group (I). Theresulting Schiff base then rearranges to produce fructose-lysinemodified proteins (II). The reactions leading up to (II) are freelyreversible. (II) can rearrange to produce 3DG and free protein lysine.Subsequent reaction between 3DG and protein is the first irreversiblestep in AGE-protein formation.

Insofar as is known, it has never been reported that 3DG can be producedby alternative pathways, or indeed, that the major source of 3-DG isfrom an enzyme catalyzed metabolic pathway, rather than from theuncatalyzed reactions shown in FIG. 1.

Diabetic patients have significantly more 3DG in serum than donon-diabetic patients (12.78±2.49 μM versus 1.94±0.17 μM). (ToshimitsuNiwa et al., Nephron, 69: 438 (1995)). Nonetheless, this toxic compoundis found in normal healthy individuals. Thus, it is not surprising thatthe body has developed a detoxification pathway for this molecule. Oneof these reactions is catalyzed by aldehyde reductase which detoxifies3DG by reducing it to 3-deoxyfructose (3DF) which is efficientlyexcreted in urine (Takahashi et al., Biochem, 34: 1433 (1995)). Anotherdetoxification reaction oxidizes 3DG to 3-deoxy-2-ketogluconic acid(DGA) by oxoaldehyde dehydrogenase (Fujii et al., Biochem. Biophys. Res.Comm., 210: 852 (1995)).

Results of studies to date show that the efficiency of at least one ofthese enzymes, aldehyde reductase, is adversely affected in diabetes.When isolated from normal rat liver, a fraction of this enzyme ispartially glycated on lysines 67, 84 and 140 and has a low catalyticefficiency when compared with the normal, unmodified enzyme (Takahaskiet al., Biochem., 34: 1433 (1995)). Since diabetic patients have higherratios of glycated proteins than normoglycemic individuals they arelikely to have both higher levels of 3DG and a reduced ability todetoxify this reactive molecule by reduction to 3DF.

The mechanism of aldehyde reductase has been studied. These studiesdetermined that this important detoxification enzyme is inhibited byaldose reductase inhibitors (ARIs) (Barski et al., Biochem., 34: 11264(1995)). ARIs are currently under clinical investigation for theirpotential to reduce diabetic complications. These compounds, as a class,have shown some effect on short term diabetic complications. However,they lack clinical effect on long term diabetic complications and theyworsen kidney function in rats fed a high protein diet. As will appearhereinbelow, this finding is consistent with the newly discoveredmetabolic pathway for lysine recovery underlying the present invention.A high protein diet will increase the consumption of fructose-lysine,which undergoes conversion into 3DG by the kidney lysine recoverypathway. The detoxification of the resulting 3DG by reduction to 3DFwill be inhibited by ARIs therapy, which consequently leads to anincrease in kidney damage, as compared to rats not receiving ARIs. Thisis because inhibition of the aldose reductase by the ARIs would reduceavailability of aldose reductase for reducing 3DG and 3DF.

The role of 3-DG in contributing to human disease has been previouslyinvestigated as will be appreciated from a review of the patentssummarized below.

U.S. Pat. No. 5,476,849 to Ulrich et al. describes a method ofinhibiting the formation of AGE-proteins using amino-benzoic acids andderivatives. These compounds presumably act by reacting with 3-DG andremoving it from the system before it can react with proteins to beginthe irreversible steps of AGE-protein formation.

U.S. Pat. Nos. 4,798,583 and 5,128,360 to Cerami et al. describes theuse of aminoguanidine to prevent AGE-protein formation anddiabetes-induced arterial wall protein cross-linking. Aminoguanidine wasshown to react with an early glycosylation product. This early productis 3DG, as defined herein. These patents do not contemplate thepossibility of inhibiting the formation of 3-DG. They focus exclusivelyon complexing this toxic molecule.

U.S. Pat. No. 5,468,777 to France et al. describes methods and agentsfor preventing the staining of teeth caused by the non-enzymaticbrowning of proteins in the oral cavity. Cysteine and cysteinederivatives are described as particularly useful in this application.

U.S. Pat. No. 5,358,960 to Ulrich et al. describe a method forinhibiting AGE-protein formation using aminosubstituted imidazoles.These compounds were shown to react with an early glycosylation product(3DG). No mention is made in this patent that a metabolic source of 3DGmay exist. This patent envisions that 3DG is made exclusively as anintermediate in the non-enzymatic browning of proteins.

U.S. Pat. No. 5,334,617 to Ulrich et al. describes amino acids useful asinhibitors of AGE-protein formation. Lysine and other bifunctional aminoacids are described as particularly useful in this regard. These aminoacids are described as reacting with the early glycosylation productfrom the reaction of glucose and proteins. It appears that the earlyglycosylation product described in this patent is 3DG.

U.S. Pat. No. 5,318,982 to Ulrich et al. describes the inhibition ofAGE-protein formation using as the inhibitory agent 1,2,4-triazoles. Theinhibitors described in this patent contain diamino-substituents thatare positioned to react with and complex 3DG. The patent describes thesecompounds as reacting with an early glycosylation product (3DG asdefined herein).

U.S. Pat. No. 5,272,165 to Ulrich et al. describes the use of2-alkylidene-aminoguanidines as inhibitors of AGE-protein formation. Theinhibitors described in this patent are said to be highly reactive with3DG. No mention is made of inhibiting the metabolic formation of 3DG inthis patent.

U.S. Pat. No. 5,262,152 to Ulrich et al. describes the use ofamidrazones and derivatives to inhibit AGE-protein formation. Thecompounds described in this patent are α-effect amines. W. P. Jencks,3rd ed., McGraw Hill, New York. Compounds of this category are known toreact with dicarbonyl compounds, e.g. 3DG.

U.S. Pat. No. 5,258,381 to Ulrich et al. describes the use of2-substituted-2-imidazolines to inhibit AGE-protein formation. Thecompounds described in this patent contain adjacent amino groups thatcan readily react with 3DG.

U.S. Pat. No. 5,243,071 to Ulrich et al. describes the use of2-alkylidene-aminoguanidies to inhibit AGE-protein formation. Thecompounds described in this patent are highly reactive with 3DG andfunction by complexing this reactive, toxic molecule.

U.S. Pat. No. 5,221,683 to Ulrich et al. describes the use ofdiaminopyridine compounds to inhibit AGE-protein formation. Thediaminopyridine compounds described as particularly useful will reactwith 3DG to form a stable, six-member ring containing complex.

U.S. Pat. No. 5,130,337 to Ulrich et al. describes the use ofamidrazones and derivatives to inhibit AGE-protein formation. Theinhibitors described in this patent are α-effect amines which, as isknow in the art, will rapidly react with 3DG and form stable complexes.

U.S. Pat. No. 5,130,324 to Ulrich et al. describes the use of2-alkylidene-aminoguanidines to inhibit AGE-protein formation. Thecompounds described in this patent function by reacting with the earlyglycosylation product resulting from the reaction of glucose withproteins (3DG).

U.S. Pat. No. 5,114,943 by Ulrich et al. describes the use ofamino-substituted pyrimidines to inhibit AGE-protein formation. Thecompounds described in this patent are said to rapidly react with anddetoxify 3DG.

None of the above-mentioned patents suggest inhibition of the metabolicformation of 3DG as a means of therapeutic intervention to preventdiabetic complications. Indeed, none of these patents even suggest theinvolvement of an enzymatic pathway in the production of 3DG.

U.S. Pat. No. 5,108,930 to Ulrich et al. describes a method fordetecting the levels of aminoguanidine in biological samples. This assayis described as having potential utility in determining kidney functionby measuring the aminoguanidine elimination time. The principal utilityintended for the assay method described in this patent is in themeasurement of tissue levels of aminoguanidine, so that doses sufficientto inhibit AGE-protein formation can be maintained in animal and humanstudies. No mention is made in this patent of using urine 3DG, 3DF orDGA ratios to determine diabetics at risk for complications.

U.S. Pat. No. 5,231,031 to Szwergold et al. describes a method forassessing the risk of diabetic-associated pathologic conditions anddetermining the efficacy of therapies for these complications. Thispatent describes the measurement of two phosphorylated compounds inerythrocytes of diabetic patients. These two compounds were notchemically identified in this patent. However, neither compound is 3DGor 3DF, whose levels are measured in urine in the diagnostic embodimentof the present invention.

Methods for monitoring metabolic control in diabetic patients bymeasurement of glycosylation end-products are known. The concentrationof glycosylated hemoglobin is known to reflect mean blood glucoseconcentration during the preceding several weeks. U.S. Pat. No.4,371,374, issued to A. Cerami et al., describes a method for monitoringglucose levels by quantitation of the degradation products ofglycosylated proteins, more specifically non-enzymatically glycosylatedamino acids and peptides, in urine. This method purports to utilize theaffinity of alkaline boronic acids for forming specific complexes withthe coplanar cis-diol groups found in glycosylation end-products toseparate and quantitate such end-products.

U.S. Pat. No. 4,761,368 issued to A. Cerami describes the isolation andpurification of a chromophore present in browned polypeptides, e.g.,bovine serum albumin and poly-L-lysine. The chromophore,2-(2-furoyl)-4(5)-2(furoyl)-1H-imidazole (FFI) is a conjugatedheterocycle derived from the condensation of two molecules of glucosewith two lysine-derived amino groups. This patent further describes theuse of FFI in a method for measuring “aging” (the degree of advancedglycosylation) in a protein sample wherein the sample “age” isdetermined by measuring the amount of the above-described chromophore inthe sample and then comparing this measurement to a standard (a proteinsample having an amount of FFI which has been correlated to the “age” ofthe sample).

Without wishing to be bound by any theory, it is believed that thepresent invention may be used to treat any glycogen storage disease.Glycogen storage diseases (glycogenoses or GSDs) are hereditarydisorders in which a patient is missing one or more of the enzymes thatinterconvert sugar and glycogen. The GSDs that are presently known areclassified as Types 0 to VII, depending on the identity of the missingenzyme or enzymes, and are also known by common names including vonGierke's disease, Pompe's disease, Forbes' disease, Andersen's disease,McArdle's disease, Hers' disease, and Tarui's disease. Fanconi'ssyndrome is also believed to be a glycogen storage disease, and, assuch, amenable to treatment with compounds of the present invention.

There is a long-standing, unfilled need in existing treatment regimensof diabetic patients for effective means to identify those at risk ofdeveloping diabetes-associated pathologic conditions, to prevent, reduceor delay the onset of such conditions by therapeutic intervention and todetermine the benefit of such therapeutic intervention. A parallel needexists in the treatment regimens of patients affected with glycogenstorage diseases, including Fanconi's syndrome.

SUMMARY OF THE INVENTION

The present invention arose from the discovery of a metabolic pathwaythat involves the enzyme-mediated conversion of FL to FL3P and producesrelatively high concentrations of 3-deoxyglucosone (3DG) in organsaffected by diabetes. Subsequent research into the biochemical functionof this newly discovered pathway tends to indicate that it has animportant role in the etiology of diabetic kidney disease. It is alsosuspected that this pathway contributes to the development of thevarious known diabetes-associated pathologic conditions.

This discovery has found practical application in the present inventionwhich, in one aspect, provides a class of compounds which have enzymeinhibitory activity and are effective to inhibit the enzymaticconversion of fructose-lysine to fructose-lysine-3-phosphate. Therelevant enzyme inhibitory activity of the compounds of the presentinvention is readily determinable by assay. The assay method comprisesproviding an aqueous solution of fructose-lysine, adenosine triphosphate(ATP), a source of fructose-lysine-3-phosphate kinase and a compound ofthe present invention in an amount sufficient to demonstrate inhibitoryactivity, subjecting the resulting solution to conditions promoting theformation of fructose-lysine-3-phosphate and adenosine diphosphate asproducts of the interaction of the above-mentioned kinase,fructose-lysine and adenosine triphosphate, and measuring the productionof at least one of such products, the compounds of the present inventionreducing the amount of such products, as compared to an aqueous solutionof the same relative amounts of fructose-lysine, adenosine triphosphateand source of fructose-lysine-3-phosphate kinase, without the additionof a compound of the present invention. The assay method just describedis also within the scope of the present invention.

According to another aspect, the present invention provides apharmaceutical preparation for preventing, reducing or delaying theonset of diabetic complications in a diabetic patient, comprising, as anactive agent, a compound of the invention, as described above, and apharmaceutically acceptable vehicle.

According to a further aspect of the present invention, there isprovided a method for preventing, reducing or delaying the onset ofdiabetic complications in a patient at risk of developing same, whichmethod comprises administering to the patient a compound of the presentinvention in an amount effective to inhibit the enzymatic conversion offructose-lysine to fructose-lysine-3-phosphate. This same method may beused for the prevention or treatment of other etiologically-similardisease states, as will be further described hereinbelow.

According to still another aspect, the present invention provides amethod for assessing a diabetic patient's risk of experiencing adiabetes-associated pathologic condition. This method comprisesadministering to the patient a source of glycated-lysine residues in anamount providing a predetermined dose of the glycated-lysine residues,and measuring the ratio of 3-deoxyglucosone to 3-deoxyfructose in abiological sample obtained from the patient, with reference to the ratioof 3-deoxyglucosone to 3-deoxyfructose in a normal subject, i.e., anon-diabetic subject or one having no clinical symptoms of diabetes. Thehigher ratio of 3-deoxyglucosone to 3-deoxyfructose in the diabeticpatient sample, in comparison to that of the asymptomatic subject isindicative that the diabetic patient is at higher risk of experiencing adiabetes-associated pathologic condition.

The present invention also provides a method for assessing the efficacyof therapeutic intervention in preventing diabetic complications. Themethod involves measuring the concentration of 3-deoxyglucosone,3-deoxyfructose and fructose-lysine in biological samples obtained froma diabetic patient, both before and after initiation of the therapeuticintervention. The sum of the 3-deoxyglucosone and 3-deoxyfructoseconcentrations are then compared to the concentration of fructose-lysinein the samples. A decrease in the sum of 3-deoxyglucosone and3-deoxyfructose concentrations relative to the fructose-lysineconcentration in the biological sample taken after initiation oftherapeutic intervention, as compared to the same concentrationsmeasured in the biological sample taken before initiation of thetherapeutic intervention, is indicative of the efficacy of thetherapeutic intervention.

As yet another aspect of the present invention, there is provided amethod for apprising a diabetic person of the potential of a foodproduct to contribute to the development of a diabetic-associatedpathologic condition. This method involves measuring the content ofglycated-lysine residues in the food product and providing thisinformation to diabetic patients, e.g., on the package of the foodproduct or in a publication intended for use by diabetics. In researchleading up to the present invention, it has been discovered thatelevated levels of 3DF in biological samples, e.g., urine, areassociated with a significant risk of developing diabetic complications.Thus, a method has been provided for assessing a diabetic patient's riskof experiencing a diabetes-associated pathologic condition based onmeasurement of the 3DF present in a biological sample of a diabeticpatient with reference to one or more predetermined baseline levels of3DF as an indicator of the likelihood that the patient will developdiabetic complications, or not.

Other related research led to the discovery of a method of reducingsusceptibility to carcinoma in a patient associated with the intake ofglycated proteins. The method comprises the administration of apharmaceutical composition which contains an active compound havinginhibitory activity for the enzymatic conversion of fructose-lysine tofructose-lysine-3-phosphate. Also embodied in the present invention is amethod of preventing, reducing, or delaying the onset of carcinomacaused by the formation of AGE-proteins. The method comprisesadministering a therapeutic amount of an agent that inhibits productionof 3-deoxyglucosone.

As a means to further assess the molecular mechanism of malignanttransformation associated with administration of a diet containingglycated proteins, a method for inducing carcinoma in a susceptible testanimal has been discovered which comprises feeding the animal with aglycated protein diet for a sufficient time period, such that3-deoxyglucosone is elevated in biological fluids at least three fold.Such animals would be assessed relative to untreated control animals.

A method of screening for substances which affect the development ofcarcinoma has also been discovered. Carcinoma will be induced in testanimals via feeding of glycated protein diet such that 3DG levels areelevated at least 3 fold in biological fluids. The animals are thendivided into two groups, one of which will receive the compound to beassessed, while the other group serves as a negative control. After asuitable time period, both groups of animals will be sacrificed and thepresence and/or absence of carcinoma in both groups assessed.

Finally, another method for screening for substances which prevent,reduce or delay the onset of carcinoma comprises the steps of feedingsusceptible test animals a glycated protein diet in an amount and for atime sufficient to maintain 3-deoxyglucosone (3DG) content of abiological fluid elevated at least 3-fold relative to the 3DG content ofa biological fluid from a similar susceptible test animal fed a dietsubstantially free of the glycated protein. A test substance will thenbe administered to one portion of the test animals but not to the otherportion. The animals will then be sacrificed and tissue sectionscompared from each such portion of susceptible test animals to assessthe effects of the test substance.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the initial step involved in the multi-step reactionleading to irreversibly-modified AGE-proteins.

FIG. 2 illustrates the reactions involved in the lysine recoverypathway.

FIG. 3 is a graphical representation of a urinary profile showing thevariation over time of 3DF, 3DG and FL from a single individual fed 2 g.of FL and followed for 24 hours.

FIG. 4 is a graphical representation of urinary excretion over time of3DF from seven volunteers fed 2 g. of fructoselysine.

FIG. 5 shows a graphical comparison of 3DF andN-acetyl-β-glucosaminidase (NAG) between a group of control animals andan experimental group maintained on a feed containing 0.3% glycatedprotein.

FIG. 6 is a graph showing the linear relationship between 3DF and 3DGlevels in urine of rats fed either a control diet or one enriched inglycated protein.

FIGS. 7A and 7B are graphical representations of fasting levels of 3DGin the urine of normals and diabetic patients plotted against thefasting level of 3DF.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are provided to facilitate understanding ofthe present invention, as described in further detail hereinbelow:

1. Glycated-Lysine Residues—The expression “glycated lysine residues”,as used herein, refers to the modified lysine residue of a stable adductproduced by the reaction of a reducing sugar and a lysine-containingprotein.

The majority of protein lysine residues are located on the surface ofproteins as expected for a positively charged amino acid. Thus, lysineresidues on proteins which come in contact with serum, or otherbiological fluids, can freely react with sugar molecules in solution.This reaction occurs in multiple stages. The initial stage involves theformation of a Schiff base between the lysine free amino group and thesugar keto-group. This initial product then undergoes the Amadorirearrangement, to produce a stable ketoamine compound.

This series of reactions can occur with various sugars. When the sugarinvolved is glucose, the initial Schiff base product will involve imineformation between the aldehyde moiety on C-1 of the glucose and thelysine ε-amino group. The Amadori rearrangement will result in formationof lysine coupled to the C-1 carbon of fructose,1-deoxy-1-(ε-aminolysine)-fructose, herein referred to asfructose-lysine or FL.

Similar reactions will occur with other aldose sugars, for examplegalactose and ribose (Dills, Am. J. Clin. Nutr., 58: S779 (1993)). Forthe purpose of the present invention, the early products of the reactionof any reducing sugar and the ε-amino residue of protein lysine areincluded within the meaning of glycated-lysine residue, regardless ofthe exact structure of the modifying sugar molecule.

Also, the terms glycated-lysine residue, glycated protein andglycosylated protein or lysine residue are used interchangeably herein,which is consistent with current usage in scientific journals where suchexpressions are often used interchangeably.

2. Fructose-lysine—The term “fructose-lysine” (FL) is used herein tosignify any glycated-lysine, whether incorporated in a protein/peptideor released from a protein/peptide by proteolytic digestion. This termis specifically not limited to the chemical structure commonly referredto as fructose-lysine, which is reported to form from the reaction ofprotein lysine residues and glucose. As noted above, lysine amino groupscan react with a wide variety of sugars. Indeed, one report indicatesthat glucose is the least reactive sugar out of a group of sixteen (16)different sugars tested (Bunn et al., Science, 213: 222 (1981)). Thus,tagatose-lysine formed from galactose and lysine, analogously to glucoseis included wherever the term fructose-lysine is mentioned in thisdescription, as is the condensation product of all other sugars, whethernaturally-occurring or not. It will be understood from the descriptionherein that the reaction between protein-lysine residues and sugarsinvolves multiple reaction steps. The final steps in this reactionsequence involve the crosslinking of proteins and the production ofmultimeric species, known as AGE-proteins, some of which arefluorescent. Proteolytic digestion of such modified proteins does notyield lysine covalently linked to a sugar molecule. Thus, these speciesare not included within the meaning of “fructose-lysine”, as that termis used herein.

3. Fructose-lysine-3-phosphate—This compound is formed by the enzymatictransfer of a high energy phosphate group from ATP to FL. The termfructose-lysine-3-phosphate (FL3P), as used herein, is meant to includeall phosphorylated fructose-lysine moieties that can be enzymaticallyformed whether free or protein-bound.

4. Fructose-lysine-3-phosphate kinase—This term refers to one or moreproteins which can enzymatically convert FL to FL3P, as defined above,when additionally supplied with a source of high energy phosphate.

5. 3-Deoxyglucosone—3-Deoxyglucosone (3DG) is the1,2-dicarbonyl-3-deoxysugar (also known as 3-deoxyhexulosone) which isformed upon breakdown of FL3P to yield free lysine and inorganicphosphate. For purposes of the present description, the term3-deoxyglucosone is intended to include all possible dicarbonyl sugarswhich are formed upon breakdown of FL3P, having the broad definition ofFL3P stated above.

6. FL3P Lysine Recovery Pathway—A lysine recovery pathway exists inhuman kidney, and possibly other tissues, which regenerates unmodifiedlysine as a free amino acid or incorporated in a polypeptide chain. Aswill be further explained below, this pathway is an important factorcontributing to the complications of diabetes.

7. AGE-Proteins—The term “AGE-proteins” (Advanced Glycation End-productmodified proteins) has been used in scientific journals, and is usedherein, to refer to the final product of the reaction between sugars andproteins (Brownlee, Diabetes Care, 15: 1835 (1992) and Niwa et al.,Nephron, 69: 438 (1995)). It is clear that the reaction, for example,between protein lysine residues and glucose does not stop with theformation of fructose-lysine. FL can undergo multiple dehydration andrearrangement reactions to produce non-enzymatic 3DG, which reacts againwith free amino groups, leading to cross-linking and browning of theprotein involved. Indeed, there is reasonable evidence that 3DG, asdefined hereinabove, is a central intermediate in this modificationreaction.

8. “Glycated Diet”—As used herein, this expression refers to any givendiet in which a percentage of normal protein is replaced with glycatedprotein. The expression “glycated diet” and “glycated protein diet” areused interchangeably herein.

At least some, and possibly all, of the complications of diabetes aredue to the covalent modification of proteins by glucose and otherreactive sugars. M. Brownlee, Diabetes, 43: 836 (1994). As noted above,diabetic humans and animals have been shown to have higherconcentrations of sugar modified proteins than normal. In fact, theincrease in diabetes-associated AGE-proteins is greater than theincrease in blood glucose levels.

Previously, it had been generally accepted that the origin of 3DG invivo was from the decomposition of proteins containing glycated lysineresidues. It had also been commonly believed that these glycated-lysinescould not be used as an amino acid source. As will appear hereinbelow,this previous belief was incorrect.

9. “Susceptible test animal”—As used herein this expression refers astrain of laboratory animals which, due to the presence of certaingenetic mutations, have a higher propensity towards malignanttransformation and tumor formation. Unless otherwise specified, the Ekerrat which has a mutation in the tuberous sclerous gene (Tsc-2) wasutilized in the studies described herein. One of ordinary skill in theart is aware of a variety of other laboratory rat or mouse strains withincreased propensity for tumor formation. The phrase “similarsusceptible test animal” refers to animals of a comparable geneticbackground which are used as control, untreated animals.

As mentioned above, the present invention evolved from the discovery ofa previously unknown metabolic pathway which produces 3DG in anenzyme-catalyzed reaction. This enzymatic pathway is capable ofenzymatic inhibition, thereby reducing the production of toxic 3DG.

During the course of a series of studies on diabetic kidneys,examination of ³¹P NMR spectra from perchloric acid extracts of kidneysfrom streptozotoxin induced diabetic rats revealed an unusual new peakin the NMR spectrum. Previous studies by the present inventors haddemonstrated the presence of fructose-3-phosphate in rat lens and humanerythrocytes (A. Petersen et al., Biochem. J., 284: 363-366 (1992); Lalet al., Arch. Biochem. Biophys., 318: 191 (1995); Szwergold et al.,Science, 247: 451 (1990) and Lal et al., Investigative Opthalmology andVisual Science, 36(5): 969 (1995)). Earlier studies had identified otherunusual phosphorylated sugars in rat lens (Szwergold et al., Diabetes,44: 810 (1995) and Kappler et al., Metabolism, 44: 1527 (1995)). Thus itwas reasonable to assume that this newly identified peak was anotherphosphorylated sugar. Further extensive laboratory investigationrevealed that this new compound was not a simple sugar, but ratherfructose-lysine phosphorylated on the 3 position of the fructosecomponent.

This identification was confirmed in two ways. Authenticfructose-lysine-3-phosphate (FL3P) was synthesized by the proceduredisclosed in Example 2, below, and shown to co-resonate in the ³¹P NMRspectrum with the peak in diabetic rat kidneys. Syntheticfructose-lysine was also injected into non-diabetic rats. These ratsshowed a substantial increase in the levels of FL3P in their kidneysfollowing this injection.

Two experiments were conducted to demonstrate that FL3P is deriveddirectly from FL in an enzyme catalyzed reaction. Fructose-lysinelabeled with deuterium at the C3 position of the fructose moiety wassynthesized and injected into rats. Three hours after injection, thekidneys of these rats were removed and extracted with perchloric acid.NMR spectroscopy revealed that the FL3P material isolated from theserats contained the deuterium label at the C3 position of the fructosemoiety. In addition, rat kidney homogenates demonstrate the ability toproduce FL3P in a reaction requiring both ATP and fructose-lysine. Thislast-mentioned experiment confirms the presence of a specific FL3Pkinase, as no FL3P is formed when only fructoselysine and ATP areincubated together under physiological conditions. Further experimentswhich involved the fractionation of kidney cortex have demonstrated thatthis kinase activity is not distributed uniformly in the kidney but isconcentrated in the proximal tubular region, which is one of theearliest anatomical sites to demonstrate damage in human and animaldiabetic kidneys.

FL3P is not stable in aqueous solution. It rapidly degrades to form 3DG,lysine and inorganic phosphate. This reaction also occurs in vivo. It isnot currently know if the degradation of FL3P in vivo is a spontaneousor enzyme catalyzed reaction. It is strongly suspected, however, thatenzymatic catalysis is involved, as the production of 3DG fromfructose-lysine occurs very rapidly in intact kidney.

The reaction steps in the FL3P lysine recovery pathway are presented inFIG. 2. In the first step, fructose-lysine and ATP react to formfructose-lysine-3-phosphate (FL3P) and ADP in a reaction catalyzed byFL3P kinase. Phosphorylation occurs on the 3-position of the fructosemoiety, leading to destabilization of the fructoselysine molecule. Theresulting FL3P then decomposes to form 3-deoxyglucosone (3DG), inorganicphosphate, and unmodified, free, reusable lysine, which is available forutilization in protein synthesis. Aldehyde reductase detoxifies 3DG byreduction to 3-deoxyfructose (3DF), which is excreted in urine.

Although FIG. 2 illustrates this pathway using the most prevalentglycated-lysine, fructose-lysine, it will be readily apparent to thoseskilled in the art that a wide variety of similar molecules can fluxthrough this pathway. Indeed, as will be explained in further detailbelow, the substrate selectivity of the FL3P lysine recovery pathway isquite broad, warranting the broad definition of the terms given above.

Additional experiments have shown that the lysine recovery pathway isfound in a wide variety of animal species, including sheep, pig, dog,rabbit, cow, mice and chicken. This pathway is also present in humans.The ubiquitous presence of the FL3P lysine recovery pathway can beunderstood, given that lysine is an essential amino acid which ispresent in relatively low concentrations in most foods. In addition, anappreciable percentage of the lysine residues in food will exist in theglycated form and the proportion of this modified lysine will increasewhen the food is cooked. Since these glycated lysine residues can not beutilized for protein synthesis, a recovery pathway for lysine is ofgreat utility and affords a selective advantage to organisms whichpossess it.

Diabetes has two effects on the lysine recovery pathway. Blood proteinscontain higher concentrations of glycated-lysines when isolated fromdiabetics than from non-diabetic individuals. Thus, diabetics aresubject to greater flux through the lysine recovery pathway thannon-diabetics. Additionally, from preliminary observations on the ratiosof 3DG and 3DF in the urine of diabetics and normals, diabetics appearto have a reduced ability to detoxify 3DG that is produced via thispathway. These two factors combine to produce higher urinaryconcentrations of 3DG in diabetics (See FIG. 7; also Lal et al., Arch.Biochem. and Biophys., 342(1): 254-60 (1997).

The agents involved in the lysine recovery pathway have been identifiedin other tissues besides kidney, specifically red blood cells, lens, andperipheral nerve tissues. All of these tissues are affected by thecomplications of diabetes. The location in red blood cells correlateswith the microvascular complications of diabetes, e.g., diabeticretinopathy, the kidney location correlates with diabetic nephropathy,while the location in peripheral nerve correlates with diabeticperipheral neuropathy. These agents are also found in pancreas.Experiments are in progress to determine the presence of these agents inskin. If found to be present, it is believed that their deleteriouseffects may be ameliorated by a topical treatment using the inhibitorycompounds of the invention in a suitable vehicle to prevent collagencrosslinking, and thereby improve skin elasticity.

Experiments have been conducted that tend to prove that humans produceboth 3DG and 3DF from orally ingested proteins containingglycated-lysine residues. These experiments, which are described indetail below, convincingly demonstrate that the lysine recovery pathwayexists in humans. These experiments also shed light on a puzzlingphenomenon, namely, that some diabetics develop diabetic complications,while others, even those in poor glycemic control, do not develop suchcomplications. The reason for this phenomenon is apparent from the datapresented herein. Diabetics have a differing ability to detoxify 3DG. Asubset of the diabetic population appears to have relatively higheraldehyde reductase activities than does the majority of diabetics.Consequently, these individuals are capable of handling the increasedflux through the lysine recovery pathway by efficiently detoxifying thehigher than normal level of 3DG. Others with impaired capacity are lessable to detoxify their elevated 3DG levels, and consequently are athigher risk of developing diabetic complications.

As will be described in more detail below, it has been experimentallydemonstrated that stimulation of the lysine recovery pathway can occurthrough the use of a glycated protein diet. As was the case with FLabove, elevation of FL3P, 3DG and 3DF was observed in test animals thatwere fed the glycated protein diet.

The enzyme inhibitor compounds of the invention block the lysinerecovery pathway, preventing formation of toxic 3DG from FL3P.

Described below is a set of extensive criteria that a suitable enzymeinhibitor should display for use in the practice of this invention, aswell as certain tests for determining if any putative inhibitor meetsthese criteria. Candidate kinase inhibitors for use in accordance withthis invention may be natural products isolated from plants ormicroorganisms. Alternatively, they may be synthetic molecules derivedfrom the rational knowledge of the enzymatic reaction and its mechanism.Inhibitors may also be synthesized by combinatorial methods.Combinatorial libraries may be generated from a random starting point.Furthermore, combinatorial methods can be utilized to generate a widevariety of compounds related to previously identified inhibitors of thetarget FL3P kinase.

Regardless of the source of the putative inhibitor, compounds that donot meet all of the criteria listed below are not considered usefultherapeutic agents capable of inhibiting the lysine recovery pathway andthereby preventing, reducing or delaying the onset of diabeticcomplications or disorders of related etiology.

1. The inhibitor should be a small molecule and readily taken up bycells. In order to meet this criteria, the inhibitor must have amolecular weight of less than 2,000 and more ideally approximately 1,000daltons or less.

2. The inhibitor must show competitive, noncompetitive, irreversible orsuicide inhibition of the FL3P kinase. If the inhibitor is a competitiveor noncompetitive inhibitor, the inhibition constant, K_(i), must beless than about 1 mM. Ideally, it must be less than 100 μM and moreideally, about 40 μM or less. If the inhibitor shows suicide or otherirreversible inhibition, this requirement for inhibition constant isrendered moot.

3. The inhibitor must be both soluble in aqueous solution and stable inaqueous solution at physiological pH. The requirement for solubility ismet only if the inhibitor, or a salt of the inhibitor, is soluble inphysiological saline or serum at a concentration equal to or greaterthan 10 μM. This stability requirement is met only if a solution ofinhibitor dissolved in physiological saline at 37° C. retains greaterthan 50% of its activity after incubation for one hour. Ideally, theinhibitor must retain greater than 50% activity upon incubation for oneday or more.

4. The inhibitor must show acceptable pharmacokinetics. That is, it mustremain at a therapeutically effective concentration for at least onehour following administration of the agent. Ideally, it should maintaineffective concentration for at least eight hours. More ideally, once perday dosing should be all that is necessary in order to maintain atherapeutic concentration of the inhibitor. This requirement does notmean that the inhibitor must be able to establish a therapeuticconcentration after the first dose. Numerous examples of successfulpharmaceuticals exist where medical efficacy is seen only upon prolongeddosing. The criterion does mean that, once an efficacious concentrationis reached, this concentration should be able to be maintained forgreater than one hour following the last administration of medication. Atest for therapeutic efficacy is described herein.

5. The inhibitor must be non-toxic. This criteria requires that theinhibitor not demonstrate human toxicity when administered at thetherapeutic dose. Ideally, toxicity should not be evident when theinhibitor is present at blood and/or target tissue levels of twice thatneeded for therapeutic effect. More ideally, there should be noappreciable toxicity at levels 6 or more times the therapeutic range.Diabetic complications can only be prevented by long term inhibitortreatment. Therefore, the requirement for non-toxicity must include bothacute toxicity and chronic toxicity that may become evident overextended, long term use. Toxicity of candidate molecules can be readilyassessed using well established animal studies. Human toxicity isassessed in stage one clinical trials.

Included among the compounds useful in the practice of this inventionare those of the formula:

wherein X is —NR′—, —S(O)—, —S(O)₂—, or —O—, R′ being selected from thegroup consisting of H, and linear or branched chain alkyl group (C₁-C₄)and an unsubstituted or substituted aryl group (C₆-C₁₀) or aralkyl group(C₇-C₁₀); R is a substituent selected from the group consisting of H, anamino acid residue, a polyaminoacid residue, a peptide chain, a linearor branched chain aliphatic group (C₁-C₈), which is unsubstituted orsubstituted with at least one nitrogen- or oxygen-containingsubstituent, a linear or branched chain aliphatic group (C₁-C₈), whichis unsubstituted or substituted with at least one nitrogen- oroxygen-containing substituent and interrupted by at least one —O—, —NH—,or —NR″— moiety, R″ being linear or branched chain alkyl group (C₁-C₆)and an unsubstituted or substituted aryl group (C₆-C₁₀) or aralkyl group(C₇-C₁₀), with the proviso that when X represents —NR′—, R and R′,together with the nitrogen atom to which they are attached, may alsorepresent a substituted or unsubstituted heterocyclic ring having from 5to 7 ring atoms, with at least one of nitrogen and oxygen being the onlyheteroatoms in said ring, said aryl group (C₆-C₁₀) or aralkyl group(C₇-C₁₀) and said heterocyclic ring substituents being selected from thegroup consisting of H, alkyl (C₁-C₆), halogen, CF₃, CN, NO₂ and—O-alkyl(C₁-C₆) R₁ is a polyol moiety having 1 to 4 linear carbon atoms,Y is a hydroxymethylene moiety —CHOH—; Z is selected from the groupconsisting of —H, —O-alkyl(C₁-C₆), -halogen —CF₃, —CN, —COOH, and—SO₃H₂, and optionally —OH; and the isomers and pharmaceuticallyacceptable salts of said compound, except that X—R in the above formuladoes not represent hydroxyl or thiol.

Illustrative examples of nitrogen- or oxygen-containing “R” substituentsinclude those derived from γ-amino-α-hydroxy butyric acid(—(CH₂)₂—CHOH—COOH), 1,2,4 triaminobutane (—(CH₂)₂—CHNH₂—CH₂NH₃),3,6-diamino-5-hydroxyheptanoic acid (—CH₂—CH(OH)—CH₂—CH(NH₂)—CH₂—COOH),and the like.

The structure of formula I has asymetric centers and may occur asracemates, racemic mixtures and various stereoisomers, all of suchisomeric forms being within the scope of this invention, as well asmixtures thereof.

Although certain of the compounds having the structure of formula I,above, were previously known, others are believed to be novel and assuch are within the scope of the present invention, as is the use of allof the compounds of formula I for inhibiting the enzyme-catalyzedproduction of 3DG in vivo.

Inhibitors of the above formula may be prepared by reacting theappropriate sugar, e.g., glucose, galactose, mannose, ribose, xylose, orthe like, with an amino- or hydroxyl-substituted reactant of the typedescribed herein in the presence of an agent, such as NaBH₃CN, thatselectively reduces the Schiff-base intermediate to an amine, therebyproducing an inhibitor having an alcohol moiety (i.e., Y═—CH(—OH)—). Thereactive moiety of an amino acid reactant, when used, may be the aminegroup on the alpha-carbon, or the amine group or hydroxyl group on theacid side chain. Suitable amino acids encompass the essential aminoacids. Specific examples include without limitation, glycine, alanine,valine, leucine, isoleucine, serine, threonine, methionine, asparticacid, phenylalanine, tyrosine, histidine and tryptophan. Other suitablereactants are from the broader class of aminocarboxylic acid, forexample, pyroglutamic acid, beta-alanine, gamma-aminobutyric acid,epsilon-amino caproic acid and the like. N-acyl derivatives of theabove-mentioned amino acids, such as formyl lysine, may also be used ifdesired.

Other appropriate reactants include, without limitation, unsubstitutedor substituted aryl(C₆-C₁₀) compounds, wherein the substituent may bealkyl(C₁-C₃), alkoxy, carboxy, nitro or halogen groups, unsubstituted orsubstituted alkanes, wherein the substituent may be at least one alkoxygroup; or unsubstituted or substituted nitrogen-containing heterocycliccompounds, wherein the substituents may be alkyl(C₁-C₃), aryl(C₆-C₁₀),alkoxy, carboxy, nitro or halogen groups. Illustrative examples of thelast-mentioned group of reactants include m-methyl-, p-methyl-,m-methoxy-, o-methoxy- and m-nitro-aminobenzenes, o- and p-aminobenzoicacids; n-propylamine, n-butylamine, 3-methoxypropylamine; morpholine andpiperdine.

Representative inhibitor compounds having the above formula are setforth in the attached Table A. Examples of known compounds that may beused as inhibitors in practicing this invention include, withoutlimitation, meglumine, sorbitol lysine and mannitol lysine. A preferredinhibitor is 3-O-methyl sorbitollysine.

It appears that the locus of uptake of the inhibitors in vivo is thekidney, as demonstrated by the data in Example 16, below. TABLE ACompound Name X R R₁ Y Z a 3-O-methyl sorbitollysine —N—H

—O—CH₃ galactitol lysine ″ ″ ″

—OH 3-deoxy sorbitol lysine ″ ″ ″ ″ —H 3-deoxy-3-fluoro- xylitol lysine″ ″

″ —F 3-deoxy-3-cyano sorbitol lysine ″ ″

″ —C≡N b 3-deoxy-sedoheptitol spermine —N—CH₃

Ha - lysine residueb - spermine residue

The inhibitor compounds described herein can form pharmaceuticallyacceptable salts with various inorganic or organic acids or bases.Suitable bases include, e.g., alkali metal salts, alkaline earth metalsalts, ammonium, substituted ammonium and other amine salts. Suitableacids include, e.g., hydrochloric acid, hydrobromic acid andmethanesulfonic acid.

The pharmaceutically acceptable salts of the compounds of formula I canbe prepared following procedures which are familiar to those skilled inthe art.

The ability of a compound to inhibit the FL3P kinase can be determinedusing a wide variety of kinase activity assays. One useful assayinvolves incubating the potential inhibitor with fructose-lysine and ATPin the presence of kidney homogenate or other enzyme source.

A solution of the assay components is prepared, which typically contains1 millimole or less of the inhibitor compound of this invention, anamount of fructose lysine (FL) in the range of 1-10 millimoles, anamount of ATP in the range of 0.1-10 millimoles and an amount of theenzyme source which is sufficient to convert FL to fructoselysine-3-phosphate. The incubation should be conducted within a pH rangeof 4.5 to 9.5 and ideally at neutral or near neutral pH. The incubationshould be carried out at a temperature that is compatible with enzymeactivity, between 40 and 40° C. Ideally, the incubation is carried outat physiological temperature. After incubation, the reaction is stoppedby acid precipitation of the protein and the production of FL3P measuredby ³¹P-NMR spectroscopy. FL3P production will be reduced or eliminatedin samples containing an inhibitor compound when compared to controlsamples that are free of inhibitor.

Other assays have utility for the rapid determination of enzymeinhibition. One such assay involves the use of fructose-lysine andγ-labelled ³²P or ³³P-ATP. Since FL3P does not bind to Dow-1 but ATP andmost other phosphates do, it is possible to separate the product FL3Pfrom the remaining reaction mixture by passing the assay solutionthrough a column of Dow-1 resin after a predetermined reaction time,typically 10 minutes. The resultant solution is added to a container ofscintillation liquid, e.g., Ecoscint A, and counted to determine theamount of radioactivity produced.

As it is difficult to obtain large quantities of human tissue, it ispreferable to use a recombinant version of the kinase that is clonedinto an expression system, such as E. Coli. The cloned kinase can bereadily obtained from the “shotgun” cloning of tissue specific cDNAlibraries. Such libraries are readily available from commercial sources.For example they may be obtained from Clontech, Palo Alto, Calif. Theshotgun cloning envisioned may be performed using the lambda cloningsystem commercially available from Stratagen, located in San Diego,Calif. This cloning kit contains detailed instructions for its use.

The pharmaceutical preparations of the present invention comprise one ormore of the compounds described above, as the active ingredient, incombination with a pharmaceutically acceptable carrier medium orauxiliary agent.

These ingredients may be prepared in various forms for administration,including both liquids and solids. Thus, the preparation may be in theform of tablets, caplets, pills or dragees, or can be filled in suitablecontainers, such as capsules, or, in the case of suspensions, filledinto bottles. As used herein, “pharmaceutically acceptable carriermedium” includes any and all solvents, diluents, or other liquidvehicle, dispersion or suspension aids, surface active agents, isotonicagents, thickening or emulsifying agents, preservatives, solid binders,lubricants and the like, as suited to the particular dosage formdesired. Representative examples of suitable carrier media includegelatine, lactose, starch, magnesium stearate, talc, vegetable andanimal fats and oils, gum, polyalkylene glycol, or the like. Remington'sPharmaceutical Sciences, Fifteenth Edition, E. W. Martin (MackPublishing Co., Easton, Pa. 1975) discloses various carriers used informulating pharmaceutical compositions and known techniques for thepreparation thereof. Except insofar as any conventional carrier mediumis incompatible with the enzyme inhibitors of the invention, such as byproducing any undesirable biological effect or otherwise interacting ina deleterious manner with any other component(s) of the pharmaceuticalpreparation, its use is contemplated to be within the scope of thisinvention.

In the pharmaceutical preparations of the invention, the active agent(s)may be present in an amount of at least 5% and generally not more than98% by weight, based on the total weight of the preparation, includingcarrier medium and/or auxiliary agent(s), if any. Preferably, theproportion of active agent varies between 65%-95% by weight of thecomposition.

Preferred supplementary active agents are compounds that bind to 3DG invivo. This class of compounds includes, without limitation,aminoguanidine, amino benzoic acid and derivatives thereof, cysteine andderivatives thereof, amino-substituted imidazoles, 1,2-disubstitutedbenzimidazoles, substituted 1,2,4-triazoles, diaminopyridine andderivatives thereof, amino-substituted pyrimidines, aminoalcohols,diamines and the like. Anti-hypertensive drugs, including particularlythe angiotensin-converting enzyme (ACE) inhibitors, may also be includedas supplementary active agents in the pharmaceutical preparations ofthis invention.

Auxiliary agents, such as compounds that will protect the activecompound from acid destruction in the stomach or facilitate theabsorption of the active compound into the bloodstream can also beincorporated into the pharmaceutical preparation, if necessary ordesirable. Such auxiliary agents may include, for example, complexingagents such as borate or other salts which partially offset the acidconditions in the stomach, and the like. Absorption can be increased bydelivering the active compound as the salt of a fatty acid (in thosecases where the active compound contains one or more basic functionalgroups).

The compounds of the invention, along with any supplementary activeingredient(s) may be administered, using any amount and any route ofadministration effective for inhibiting the FL3P lysine recoverypathway. Thus, the expression “therapeutically effective amount”, asused herein, refers to a nontoxic but sufficient amount of the enzymeinhibitor to provide the desired therapy to counteract diabeticcomplications or to inhibit the metabolic production of 3DG for othermedical reasons, such as reducing the effects of aging or other humandisease states where AGE-Protein formation has a causative role. Theexact amount required may vary, depending on the species, age, andgeneral condition of the patient, the nature of the complications, theparticular enzyme inhibitor and its mode of administration, and thelike.

The compounds of the invention are preferably formulated in dosage formfor ease of administration and uniformity of dosage. Dosage unit form asused herein refers to a physically discrete unit of enzyme inhibitorappropriate for the patient to be treated. Each dosage should containthe quantity of active material calculated to produce the desiredtherapeutic effect either as such, or in association with the selectedpharmaceutical carrier medium. Typically, the compounds of the inventionwill be administered in dosage units containing from about 1 mg to about2,500 mg of the compound, by weight of the preparation, with a range ofabout 5 mg to about 250 mg being preferred.

The compounds of the invention may be administered orally, parenterally,such as by intramuscular injection, intraperitoneal injection,intravenous infusion or the like, depending on the nature of thediabetic complication being treated. The compounds of the invention maybe administered orally or parenterally at dosage levels of about 0.7 μgto about 20 mg and preferably from about 30 μg to about 3.5 mg/kg, ofpatient body weight per day, one or more times a day, to obtain thedesired therapeutic effect.

Orally active enzyme inhibitors are particularly preferred, provided theoral dose is capable of generating blood and/or target tissue levels ofthe inhibitor that are therapeutically active. Those skilled in the artcan readily measure the levels of a small molecule inhibitor indeproteinized samples of blood, kidney and other target tissues. Theconcentration of inhibitor in these samples can be compared with thepredetermined inhibitory constant. Tissue levels that are far below theinhibitory constant suggest a lack of therapeutic activity. In the caseof irreversible inhibitors, this lack can be confirmed or refuted byassay of the FL3P kinase levels in the respective tissue. In all cases,therapeutic activity can be assessed by feeding the human or animalsubject a food rich in glycated lysine residues or fructose-lysine andmeasuring the amount of 3DG and 3DF in their urine, both before andafter feeding. Subjects that have therapeutically active inhibitor intheir systems will experience decreased secretion of both 3DG and 3DFand increased urinary secretion of fructose-lysine when compared tolevels secreted by these same subjects prior to inhibitor therapy aswill be described in further detail hereinbelow.

The compounds of the invention will typically be administered once perday or up to 4-5 times per day, depending upon the exact inhibitorchosen. While a dosing schedule of once-a-day is preferred, diabeticpatients are accustomed to paying close attention to their diseasestate, and so will readily accept more frequent dosing schedules ifrequired, so as to deliver the above-mentioned daily dosage. However,the exact regimen for administration of the compounds and compositionsdescribed herein will necessarily be dependent on the needs of theindividual patient being treated, the type of treatment administered andthe judgment of the attending physician. As used herein, the term“patient” includes both humans and animals.

The inhibitor compounds described herein are useful in counteractingdiabetic complications, especially diabetic nephropathy which affectsgreater than forty percent of diabetics and is the primary cause of endstage renal disease requiring dialysis and transplantation. In addition,these inhibitors may be used for the prevention or treatment of otherpathological conditions attributable to the formation of AGE-proteins,such as hypertension, stroke, neurodegenerative disorders, e.g., seniledementia of the Alzheimers type, circulatory disease, glycogen storagediseases including Fanconi's syndrome, atherosclerosis, osteoarthritis,cataracts and the general debilitating effects of aging.

Preliminary experiments have shown that serious adverse health effectsresult from stimulation of the lysine recovery pathway through long-termconsumption of glycated proteins. As was the case with FL, elevation ofFL3P, 3DG and 3DF was observed in test animals that were fed a glycatedprotein diet. See Table B. After eight months of such a diet clearevidence of kidney pathology, resembling that found in diabetic kidneys,was seen in the animals on the glycated protein diet, as describedfurther in Example 10, below. Transient elevation of 3DG and 3DF levelswere also observed in the urine of human volunteers who ate a smallamount of the glycated protein. TABLE B % Glycated FL3P conc. 3DG/3DFconcs Protein (nM-in Kidney) (μM-in plasma) 0 97 1.4/0.05 1 295 — 2.5605 — 5 937 — 10 1066 3.6/0.12 20 1259 5.2/0.14 30 1267 6.2/0.28

Since stimulation of the newly discovered lysine recovery pathway leadsto substantial increases in systemic 3DG levels, an investigation wascarried out to determine whether a glycated diet would cause significanteffects on pregnancy. The results obtained so far suggest there is avery strong effect due to this pathway, as will appear in the examplesthat follow.

Furthermore, it is well known that in susceptible strains of rats andmice the diets on which the animals are maintained in early life(following weaning), can have a marked effect on the incidence of type 1diabetes, with the incidence ranging from 10% to 90%. Considerableeffort has been put into investigating this phenomenon over the last 10years. See, for example, Diabetes, 46(4): 589-98 (1997) and DiabetesMetab. Rev., 12(4): 341-59 (1996), and references cited therein. Aninvestigation has been undertaken by certain of the present inventorswith respect to two diets which are at the extremes for induction ofdiabetes. AIN-93 (Dyets, Inc.) causes the least incidence of diabetesand produces the lowest ratio of urinary 3DF/creatinine (1.0) yetobserved. Purina 500 induces the highest incidence of diabetes andproduces a 2.5 fold increase in the 3DF/creatinine ratio. Since FL3P,3DG and 3DF were observed in the pancreas of rats, it is likely thatfructoselysine kinase and the metabolites of this metabolic pathway areinvolved in the development of Type I diabetes. Animals which aresusceptible to this type of diabetes (a useful model of insulindependent or Type I diabetes in humans) have an abnormal immune systemwhich makes them sensitive to an unknown antigen which develops in theβ-cells of the pancreas, resulting in an autoimmune attack by theanimal's own immune system on its β-cells. This results in theirsubsequent destruction, thereby depriving the animal of the ability tomake insulin. It is well known that 3DG reacting with proteins can makenew antigenic sites. Thus, the source of the antigenic properties of thevarious diets appears to be the 3DG created by the decomposition offructoselysine-3-phosphate in the pancreas.

Also, because 3DG is known to interact with amines generally, it may beable to interact with DNA and show mutagenic and carcinogenic potential,as well as crosslink proteins.

The discovery of the FL3P lysine recovery pathway makes it practical,for the first time, to differentiate the diabetic population and todetermine which subset of the population is likely to develop todiabetic complications. This determination can be conveniently carriedout on a biological fluid of the test subject, such as urine, bloodfractions (particularly plasma or serum), lymph fluid, interstitialfluid or the like.

After an overnight fast, a human subject is fed a food source containinga relatively high concentration of glycated-lysine residues. By way ofexample, this food can be in the form of a casein/sugar “cookie”, suchas described in Example 5, below, or some other suitable source ofglycated-lysines or synthetic fructose-lysine. When proteins containingglycated-lysine residues are utilized, the content of glycated-lysineshould be preferably between 0.02 and 10% of total protein amino acid,or more preferably between about 0.2 and 0.4%. The total amount ofglycated-lysine residues in the oral dose should be about 0.3 grams.Preferably, a urine sample is collected before consumption of theglycated-lysine source, then at one, three and five hours, or such otherappropriate times as may be warranted by the individual clinicalsituation.

The 3DG and 3DF levels in these urine samples are measured and theratios of these metabolites calculated. The particular methodologyutilized in this measurement is not essential to the practice of thisinvention. The GC method described in Example 5, below, may be utilized,if desired. Alternatively, calorimetric or immunological assay methodscan be used, as will be apparent to those skilled in the art.

It is clear that the major risk factor faced by diabetics is glycemiccontrol, as was clearly demonstrated by the recently completed DiabetesControl and Complications Trial. However, the incidence of diabeticcomplications cannot be explained solely by blood sugar levels; a fairlywide scatter is seen when the incidence of diabetic complications iscompared to historical blood sugar levels.

One method for determining that subset of the diabetic population whichis most at risk for developing diabetic complications is a particularlysignificant aspect of the present invention. This method involves themeasurement of FL, 3DG and 3DF levels before and, optimally, afteringesting a source of glycated lysine.

For example, normal subjects have a fasted 3DG to 3DF ratio in urine ofabout 0.025, whereas diabetics have higher ratios, which may be up tofive fold higher, or more. This is borne out by the data in FIG. 7,which shows that normoglycemics have a 3DG/3DF ratio of 0.025 (1/39.77)with quite tight scatter around this value, whereas diabetics have amore than 2 fold higher average ratio (average 0.069) with much morescatter around the average.

As demonstrated herein, diabetics have increased production of 3DG.Therefore, resistance to diabetic complications requires highlyefficient removal of this toxic metabolite. The ratio of 3DG to 3DF,calculated by the method described herein, allows one to assess theefficiency of the 3DG detoxification pathways. Those individuals withlow ratio will be generally resistant to developing diabeticcomplications. Individuals with higher ratios, including ratioscontained within the normal range, are more at risk, while individualswith elevated ratios above the normal range are particularly at risk fordeveloping these complications.

Recent measurements of fructoselysine (FL) in the plasma and urine offour different rat strains have demonstrated considerable variability inthe manner in which their respective kidneys process FL in blood. In twoof the four strains (Long Evans, Brown Norway) virtually all of the FLfiltered by the kidney appeared in the urine based upon ratios of thiscompound and its metabolites with creatinine. With the other two strains(Sprague Dawley, Fischer) 10-20% of the FL in the plasma appeared in theurine, based on comparisons with creatinine filtration. Thesemeasurements strongly suggest a major variability in FL processing inthe mammalian kidney. Given what is known about the functionalequivalence of rodent and human kidneys, it is reasonable to assume asimilar variation in FL processing will exist among humans. Since FL isthe primary input to the fructoselysine recovery pathway, the entirepathway is likely to be substantially stimulated in those humans in whoma large amount of FL is absorbed from the ultrafiltrate, leading to thehigh local levels of 3-deoxyglucosone (3DG) in the kidney, as well assystemically throughout the body. This observation may serve as thebasis of a diagnostic test in which the comparison of a sample of plasmaor serum contemporaneously obtained with a urine sample would determinethe flux of FL into the kidney, and the fraction of that flux whichappears in the urine. Those individuals in whom this ratio issubstantially lower than one (1) would then be at risk for developing avariety of kidney pathologies including, but not limited to, diabeticnephropathy, kidney failure in old age and kidney carcinoma.

Therapeutic efficacy of the kinase inhibitors of the invention can beeasily and safely determined using a test of the lysine recoverypathway. The test protocol is identical to the one presented immediatelyabove, with the exception that urinary fructoselysine levels aremeasured in addition to urinary 3DG and 3DF levels. It is useful toconduct this test both before and after initiating FL3P kinase inhibitortherapy. The urine levels of 3DG and 3DF are summed at each time pointand compared to the levels of fructose-lysine measured in the samesample.

The peak levels of 3DG and 3DF found in urine following ingestion offood rich in glycated-lysine residues are derived from the activity ofthe lysine recovery pathway. The ratio of the concentration of thesemetabolites to unreacted fructose-lysine (which is a normal component ofhuman urine) reflects the activity of this pathway. Inhibition of thelysine recovery pathway will cause a decrease in the amount of 3DG and3DF excreted, and an increase in the excreted levels of fructose-lysine.Thus, therapeutic efficacy of a kinase inhibitor can be quantitated bymeasuring the decrease of the (3DG+3DF)/fructose-lysine ratio followinginitiation of therapy. It is noteworthy that urine volume or metaboliteconcentrations are not a factor in interpreting this assay, as only aratio of metabolites is considered.

It will be appreciated from the foregoing disclosure that orallydigested food containing high concentrations of glycated-lysine residueswill lead to the production of kidney and serum 3DG. It is reasonable tocaution individuals at risk for kidney disease, for example diabetics,to avoid food with these high concentrations. Concentrations ofglycated-lysine residues can be measured using a wide variety ofmethods. One such measurement method is described in Example 4, below.However, any suitable measurement methodology that accurately determinesthe levels of glycated-lysine residues can be substituted in place ofthe assay method exemplified below. Examples of assay methodsspecifically contemplated include but are not limited to calorimetricand immunological methods.

Regardless of the method of measurement employed, it is within the scopeof the present invention to determine the content of glycated-lysineresidues in prepared foods and to apprise individuals at risk fordeveloping kidney dysfunction of these determinations, so that suchindividuals may refrain from ingesting foods high in glycated-lysinecontent.

The following examples are provided to describe the invention in furtherdetail. These examples are provided for illustrative purposes only, andshould in no way be construed as limiting the invention. Alltemperatures given in the examples are in degrees centigrade unlessotherwise indicated.

EXAMPLE 1 Isolation and Identification of FL3P

A ³¹P NMR analysis of a perchloric acid extract of diabetic rat kidneysshowed a new sugar monophosphate resonance at 6.24 ppm which is notobserved in non-kidney tissue and is present at greatly reduced levelsin non-diabetic kidney. The compound responsible for the observedresonance was isolated by chromatography of the extract on amicrocrytalline cellulose column using 1-butanol-acetic acid-water(5:2:3) as eluent. The structure was determined by proton 2D COSY to befructose-lysine 3-phosphate. This was later confirmed by injectinganimals with FL, prepared as previously described (Finot and Mauson,Helv. Chim. Acta, 52: 1488 (1969)), and showing direct phosphorylationto FL3P. Using FL specifically deuterated in position-3 confirmed theposition of the phosphate at carbon-3. This was performed by analyzingthe ³¹P NMR spectra both coupled and decoupled. The normal P—O—C—Hcoupling produces a doublet in FL3P with a J value of 10.3 Hz, whereasP—O—C-D has no coupling and produces a singlet both coupled anddecoupled, as was found for 3-deuterated FL3P. A unique property of FL3Pis that when treated with sodium borohydride it is converted into twonew resonances at 5.85 and 5.95 ppm, which correspond to mannitol andsorbitol-lysine 3-phosphates.

EXAMPLE 2

SYNTHESIS OF FL3P: 1 mmol of dibenzyl-glucose 3-phosphate and 0.25 mmolof α-carbobenzoxy-lysine was refluxed in 50 ml of MeOH for 3 hours. Thesolution was diluted with 100 ml water and chromatographed on a Dow-50column (2.5×20 cm) in the pyridinium form and eluted first with water(200 ml) and then with 600 ml buffer (0.1M pyridine and 0.3M aceticacid). The target compound eluted at the end of the water wash and thebeginning of the buffer wash. Removal of the cbz and benzyl blockinggroups with 5% Pd/C at 20 psi of hydrogen gave FL3P in 6% yield.

EXAMPLE 3

ENZYMATIC PRODUCTION OF FL3P FROM FL AND ATP AND ASSAY FOR SCREENINGINHIBITORS: Initially ³¹P NMR was used to demonstrate kinase activity inthe kidney cortex. A 3 g. sample of fresh pig kidney cortex washomogenized in 9 ml. of 50 mM Tris.HCl containing 150 mM KCl, 5 mM DTT,15 mM MgCl₂, pH 7.5. This was centrifuged at 10,000 g for 30 minutes,and then the supernate centrifuged at 100,000 g for 60 minutes. Ammoniumsulfate was added to 60% saturation. After 1 hour at 4° the precipitatewas collected by centrifugation and dissolved in 5 ml. of originalbuffer. A 2 ml aliquot of this solution was incubated with 10 mM ATP and10 mM of FL (prepared as in Example 1, above) for 2 hours at 37°. Thereaction was quenched with 300 uL of perchloric acid, centrifuged toremove protein, and desalted on a column of Sephadex G 10 (5×10 cm). ³¹PNMR analysis of the reaction mixture detected formation of FL3P.

Based on the proof of kinase activity thus obtained, a radioactive assaywas developed. This assay was designed to take advantage of the lack ofbinding to Dow-1 anion exchange resin by FL3P. This characteristic ofFL3P was discovered during efforts to isolate it. Since most phosphatesbind to this resin, it was suspected that the bulk of all compounds thatreact with ATP as well as any excess ATP would be bound, leaving FL3P insolution. The first step was to determine the amount of resin requiredto remove the ATP in the assay. This was accomplished by pipetting themixture into a suspension of 200 mg. of Dow-1 in 0.9 ml H₂O, vortexingand centrifuging to pack the resin. From this 0.8 ml. of supernate waspipetted onto 200 mg. of fresh dry resin, vortexed and centrifuged. A0.5 ml volume of supernate was pipetted into 10 ml of Ecoscint A andcounted. Residual counts were 85 cpm. This procedure was used for theassay. The precipitate from 60% ammonium sulfate precipitation of thecrude cortex homogenate was redissolved in the homogenate buffer at 40.The assay contains 10 mM γ³³P-ATP (40,000 cpm), 10 mM FL, 150 mM KCl, 15mM MgCl₂, 5 mM DTT in 0.1 ml of 50 mM Tris.HCl, pH 7.5. The relationshipbetween rates of FL3P production and enzyme concentration was determinedusing triplicate determinations with 1, 2 and 4 mg of protein for 30minutes at 37°. Blanks run concurrently without FL were subtracted andthe data recorded. The observed activity corresponds to an approximateFL3P synthesis rate of 20 nmols/hr./mg. protein.

EXAMPLE 4 Inhibition of the Formation of 3-Deoxyglucosone by Meglumineand Various Polyollysines

a. General Polyollysine Synthesis.

The sugar (11 mmoles), α-carbobenzoxy-lysine (10 mmols) and NaBH₃CN (15mmoles) were dissolved in 50 ml of MeOH—H₂O (3:2) and stirred at 25° for18 hours. The solution was treated with an excess of Dow-50 (H) ionexchange resin to decompose excess NaBH₃CN. This mixture (liquid plusresin) was transferred onto a Dow-50 (H) column (2.5×15 cm) and washedwell with water to remove excess sugar and boric acid. Thecarbobenzoxy-polyollysine was eluted with 5% NH₄ OH. The residueobtained upon evaporation was dissolved in water-methanol (9:1) andreduced with hydrogen gas (20 psi) using a 10% palladium on charcoalcatalyst. Filtration and evaporation yields the polyollysine.

b. Experimental Protocol for Reduction of Urinary and Plasma3-Deoxyglucosone by Sorbitollysine, Mannitollysine and Galactitollysine.

Urine was collected from six rats for three hours. A plasma sample wasalso obtained. The animals were then given 10 μmols of eithersorbitollysine, mannitollysine, or galactitollysine by intraperitonealinjection. Urine was collected for another three hours, and a plasmasample obtained at the end of the three hours.

3-deoxyglucosone was measured in these samples, as described in Example5, below, and variable volumes were normalized to creatinine. Theaverage reduction of urinary 3-deoxyglucosone was 50% by sorbitollysine,35% by mannitollysine and 35% by galactitollysine. Plasma3-deoxyglucosone was reduced 40% by sorbitollysine, 58% by mannitolysineand 50% by galactitollysine.

c. Use of meglumine to reduce urinary 3-deoxyglucosone. Three rats weretreated as in b), immediately above, except meglumine (100 μmols) wasinjected intraperitoneally instead of the above-mentioned lysinederivatives. Three hours after the injection the average3-deoxyglucosone concentrations in the urine were decreased 42%.

EXAMPLE 5 Elevation of Urinary FL, 3DG and 3DF IN Humans FollowingIngestion of Glycated Protein

a. Preparation of glycated protein containing food product: 260 g. ofcasein, 120 g. of glucose and 720 ml. of water were mixed to give ahomogeneous mixture. This mixture was transferred to a metal plate andcooked at 65° for 68 hours. The resulting cake was then pulverized to acoarse powder.

This powder contained 60% protein as determined by the Kjeldahlprocedure.

b. Measurement of glycated lysine content: 1 g of the powder prepared asin step a., above, was hydrolyzed by refluxing with 6N HCl for 20 hours.The resulting solution was adjusted to pH 1.8 with NaOH solution anddiluted to 100 ml. The fructoselysine content was measured on an aminoacid analyzer as furosine, the product obtained from acid hydrolysis offructoselysine. In this way, it was determined that the cake contained5.5% (w/w) fructoselysine.

c. Experimental protocol: Volunteers spent two days on afructoselysine-free diet and then consumed 22.5 g of the food productprepared as described herein, thus effectively receiving a 2 g. dose offructoselysine. Urine was collected at 2 hour intervals for 14 hours anda final collection was made at 24 hours.

d. Measurement of FL, 3DG and 3DF in urine: FL was measured by HPLC witha Waters 996 diode Array using a Waters C18 Free Amino Acid column at46° and a gradient elution system of acetonitrile-methyl alcohol-water(45:15:40) into acetonitrile-sodium acetate-water (6:2:92) at 1 ml./min.Quantitation employed an internal standard of meglumine.

3DF was measured by HPLC after deionization of the sample. Analyses wereperformed on a Dionex DX-500 HPLC system employing a PA1 column (Dionex)and eluting with 32 mM sodium hydroxide at 1 ml./min. Quantitation wasperformed from standard curves obtained daily with synthetic 3DF.

3DG was measured by GC-MS after deionization of the sample. 3DG wasderivatized with a 10-fold excess of diaminonaphthalene in PBS. Ethylacetate extraction gave a salt free fraction which was converted to thetrimethyl silyl ethers with Tri-Sil (Pierce). Analysis was performed ona Hewlett-Packard 5890 selected ion monitoring GC-MS system. GC wasperformed on a fused silica capillary column (DB-5,25 mx.25 mm) usingthe following temperature program: injector port 250°, initial columntemperature 150° which is held for 1 minute, then increased to 290° at16°/minute and held for 15 minutes. Quantitation of 3DG employedselected ion monitoring using an internal standard of U-13C-3DG.

The graph shown in FIG. 3 represents production of FL, 3DF and 3DG inthe urine of one volunteer after consuming the glycated protein. Therapid appearance of all three metabolites is clearly evident. Both 3DFand 3DG show a slight elevation even after twenty-four hours.

The graph shown in FIG. 4 represents the formation of 3DF in each of themembers of a seven person test group. A similar pattern was seen in allcases. As appears in FIG. 4, 3DF excretion peaks about 4 hours after theFL bolus and a slight elevation of 3DF is noticeable even 24 h after thebolus.

EXAMPLE 6 Feeding Experiment

N-acetyl-β-glucosaminidase (NAGase) is an enzyme excreted into the urinein elevated concentration in diabetics. It is thought to be an earlymarker of tubular damage, but the pathogenesis of increased NAGase inurine is not well understood. The increased urinary output of NAGase indiabetics has been proposed to be due to activation of lysosomes inproximal tubules induced by diabetes with an increased output into theurine rather than destruction of cells.

The results obtained in this example show that in all comparisons 3DFand NAGase levels are elevated in the experimental group relative to thecontrol. Thus, animals fed glycated protein excrete excess NAGase intotheir urine, similar to results obtained with diabetics. There is anapproximate 50% increase in NAGase output compared with control animals.These animals also have a five-fold increase in urine 3DF compared withcontrols. Urinary 3DF correlates extremely well with 3DG, as can be seenin FIGS. 5 and 6. Both compounds appear to be removed from the plasma atthe glomerular filtration rate, with no reabsorption.

EXAMPLE 7 SDS Gel of Kidney Proteins

Two rats were injected daily with 5 μmols. of either FL or mannitol(used as a control) for 5 days. The animals were sacrificed and thekidneys removed and dissected into the cortex and medulla. Tissues werehomogenized in 5 volumes of 50 mM Tris.HCl containing 150 mM KCl, 15 mMMgCl₂ and 5 mM DTT, pH 7.5. Cellular debris was removed bycentrifugation at 10,000 g for 15 minutes, and the supernate was thencentrifuged at 150,000 g for 70 minutes. The soluble proteins wereanalyzed by SDS PAGE on 12% polyacrylamide gels as well as on 4-15 and10-20% gradient gels. In all cases, lower molecular weight bands weremissing or visually reduced from the kidney extract of the animalinjected with FL when compared with the animal injected with mannitol.

EXAMPLE 8 Synthesis of 3-O-Methylfructose Lysine

A suspension of 19.4 g (0.1 mol) of anhydrous 3-O-methyl glucose and 1 gof sodium bisulfite in 30 ml of methanol and 15 ml of glycerol wasrefluxed for 30 minutes, followed by the addition of 0.035 mol ofα-carbobenzoxy-lysine and 4 ml of acetic acid. This solution wasrefluxed for 3 hours. The solution was treated with 1 volume of waterand chromatographed on a Dowex-50 column (4×50 cm) in the pyridiniumform, and eluted first with water and then with pyridinium acetate.Fractions containing the pure material were combined and evaporated. Theresulting material was dissolved in 50 ml of water-methanol (9:1) andreduced with hydrogen gas (20 psi) using a 10% palladium on charcoalcatalyst. Filtration and evaporation gave 3-O-methyl-fructoselysine.

Other specific compounds having the structure of formula (I), above, maybe made e.g. by glycation of a selected nitrogen- or oxygen-containingstarting material, which maybe an amino acid, polyaminoacid, peptide orthe like, with a glycating agent, such as fructose, which may bechemically modified, if desired, according to procedures well know tothose skilled in the art.

EXAMPLE 9 Additional Assay for FL3P Kinase Activity

a. Preparation of Stock Solutions:

An assay buffer solution was prepared which was 100 mM HEPES pH 8.0, 10mM ATP, 2 mM MgCl₂, 5 mM DTT, 0.5 mM PMSF. A fructosyl-spermine stocksolution was prepared which was 2 mM fructosyl-spermine Hcl. A sperminecontrol solution was prepared which was 2 mM spermine Hcl.

b. Synthesis of Fructosyl-spermine: Synthesis of fructosyl-spermine wasperformed by an adaptation of a known procedure (J. Hodge and B. Fisher,Methods Carbohydr. Chem., 2: 99-107 (1963)).

A mixture of spermine (500 mg), glucose (500 mg) and sodium pyrosulfite(80 mg) was prepared in a molar ratio of 8:4:1(spermine:glucose:pyrosulfite) in 50 ml of methanol-water (1:1) andrefluxed for 12 hours. The product was diluted to 200 ml with water andloaded onto a DOW-50 column (5×90 cm). The unreacted glucose was removedby 2 column volumes of water and the product and unreacted spermine wereremoved with 0.1 M NH₄OH. Pooled peak fractions of the product werelyophilized and concentration of fructosyl-spermine was determined bymeasuring the integral of the C-2 fructosyl peak in a quantitative ¹³CNMR spectrum of the product (NMR data collected with a 45° pulse, a 10second relaxation delay and without NOE decoupling).

c. Assay of Kinase for Purification:

An incubation mixture was prepared including 10 μl of the enzymepreparation, 10 μl of assay buffer, 1.0 μCi of ³³P ATP, 10 μl offructosyl-spermine stock solution and 70 μl of water and incubated at37° C. for 1 hour. At the end of the incubation 90 μl (2×45 μl) of thesample is spotted onto two 2.5 cm diameter cellulose phosphate disks(Whatman P-81) and allowed to dry. The disks were washed extensivelywith water. After drying, the disks were placed in scintillation vialsand counted.

Each enzyme fraction was assayed in duplicate with an appropriatespermine control.

EXAMPLE 10 Kidney Pathology Observed in Test Animals on Glycated ProteinDiet

Three rats were maintained on a glycated protein diet (20% totalprotein; 3% glycated) for 8 months and compared to 9 rats of the sameage maintained on a control diet. The primary finding was a substantialincrease in damaged glomeruli in the animals on the glycated diet.Typical lesions observed in these animals were segmental sclerosis ofthe glomerular tuft with adhesion to Bowman's capsule, tubularmetaplasia of the parietal epithelium and intestitial fibrosis. Allthree of the animals on the glycated protein diet, and only one of theanimals on the control diet showed more than 13% damaged glomeruli. Theprobablity of this happening by chance is less than 2%. In addition tothe pathology observed in the glomeruli, a number of hylinated castswithin tubules were observed. More of these were found in animals on theglycated diet, although these were not quantitated. Increased levels ofNAGase were also observed in the animals on the glycated diet.

From the results of this experiment, the glycated diet appeared to causethe test animals to develop a series of histological lesions similar tothose seen in the diabetic kidney.

EXAMPLE 11 Effects of Glycated Diets on Pregnancy

In a preliminary experiment, 5 mice pairs were placed on a glycated diet(18% total protein; 3% glycated) and bred six times over a period of 7months. The resulting six pregnancies produced the following live pups;17, 23, 13, 0, 3 and 0. In view of this sharp drop in live pups afterthe third breeding, two cohorts of ten pairs each were put on either aglycated diet (13% total protein; 3% glycated) or a control diet (13%total protein; 0% glycated). Thus far, the two groups of pups have beenbred four times obtaining similar results in both groups. The firstpregnancy produced 49/20 (glycated/control) pups; the second, 18/41; thethird 37/27; and the fourth 20/33. The fifth pregnancy is currentlyunderway. The mice pairs have been tested for hyperglycemia. The bloodglucose levels are 120 and 112 mg/dl in the experimental and controlgroups, respectively.

Preliminary measurements of the 3DF levels in the mice urine indicate,as expected, a substantial elevation (approximately 5-10 fold) of thesystemic 3DF when on the glycated diet described herein.

EXAMPLE 12 Carcinogenic Effects of Fructoselysine Pathway

To investigate the carcinogenic potential of metabolites formed in thefructoselysine pathway, experiments have been conducted on a strain ofrats with a high susceptibility to kidney carcinomas. Four rats were puton a glycated protein diet and three rats on a control diet. After tenweeks on the diet, the animals were sacrificed and their kidneysexamined. In all four animals on the diet, kidney carcinomas of sizegreater than 1 mm were found, whereas no lesions this large were foundin the control animals. The probability of this happening by chance isless than 2%. The data show that the elevated 3DG levels caused by theexcess fructoselysine coming from the glycated protein in the animalsdiet found in the kidney tubular cells (known to be the cell of originof most kidney carcinomas) can interact with the cellular DNA leading toa variety of mutogenic and ultimately carcinogenic events. Thepossibility exists that this process is important in the development ofhuman cancers in the kidney and elsewhere.

EXAMPLE 13 Dietary Effects of Glycated Protein Diet on Renal CellCarcinoma in Susceptible Rats

In addition experiments assessing the relationship between a glycatedprotein diet and renal cell carcinoma, twenty-eight rats with a mutationmaking them susceptible to the development of kidney carcinoma weredivided into two cohorts. One cohort was fed a glycated protein diet:the other cohort was on a control diet. The glycated protein dietconsisted of a standard nutritious diet to which 3% glycated protein hadbeen added. The glycated protein was made by mixing together casein andglucose (2:1), adding water (2× the weight of the dried material), andbaking the mixture at 60° for 72 hours. The control was prepared in thesame way except that no water was used and the casein and glucose werenot mixed prior to baking. Rats were placed on the diets immediatelyfollowing weaning at three weeks of age and maintained on the diets adlibitum for the next 16 weeks. The animals were then sacrificed, thekidneys fixed and hemotoxylin and eosin sections were made. These wereexamined for lesions by a trained pathologist. Four types of lesionswere identified. These included: cysts, very small collections oftumor-like cells, typically less than 10 cells; small tumors, 0.5 mm orless, and tumors greater than 0.5 mm. For every type, more lesions wereobserved in the animals on the glycated diet than on the control diet asshown in the following table. CYSTS ≦10 CELLS ≦0.5 mm >0.5 mm TOTALCONTROL 2 9 9 3 23 GLYCATED 9 21 32 6 68

To summarize the results, the average number of lesions per kidneysection was computed for each diet. These were 0.82±0.74 and 2.43±2.33in the control and glycated diet, respectively. The likelihood of thishappening by chance is about 2 in 100,000.

These results provide strong support for the premise that the effects ofthe lysine recovery pathway, the discovery of which underlies thepresent invention, extend to causing mutations, and thus produce acarcinogenic effect as well. These results provide a basis for thedevelopment of therapeutic methods and agents to inhibit this pathway inorder to reduce cancer in the kidney as well as in other organs wherethis pathway may have similar effects.

EXAMPLE 14 Urinary Excretion of 3-Deoxy-Fructose is Indicative ofProgression to Microalbuminuria in Patients with Type I Diabetes

As set forth hereinabove, serum levels of the glycation intermediate,three deoxy-glucosone (3DG) and its reductive detoxification product,three deoxy-fructose (3DF), are elevated in diabetes. The relationshipbetween baseline levels of these compounds and subsequent progression ofmicroalbuminura (MA) has been examined in a group of 39 individuals froma prospective cohort of patients at the Joslin Diabetes Center withinsulin-dependent diabetes mellitus (IDDM) and microalbuminuria (basedon multiple measurements during the two years of baseline startingbetween 1990-1993) and not on ACE inhibitors.

Baseline levels of 3DF and 3DG in random spot urines were measured byHPLC and GC-MS. Individuals that progressed to either a higher level ofMA or proteinuria in the next four years (n=24) had significantly higherbaseline levels of log3DF/urinary creatinine ratios compared tonon-progressors (n=15) (p=0.02). Baseline levels determined in thisstudy were approximately 0.24 μmole/mg of creatinine in the progressorsvs. approximately 0.18 μmole/mg of creatinine ratios in thenon-progressors. Baseline 3DG/urine creatinine ratios did not differbetween the groups. Adjustment of the baseline level of HgA_(1c) (themajor fraction of glycoslyated hemoglobin) did not substantially alterthese findings. These results provide additional evidence of theassociation between urinary 3DF and progression of kidney complicationson diabetes.

A. Quantification of 3-Deoxyfructose

Samples were processed by passing a 0.3 mL aliquot of the test samplethrough an ion-exchange column containing 0.15 mL of AG 1-X8 and 0.15 mLof AG 50W-X8 resins. The columns were then washed twice with 0.3 mLdeionized water, aspirated to remove free liquid and filtered through a0.45 mm Millipore filter.

Injections (50 μL) of the treated samples were analyzed using a DionexDX 500 chromatography system. A carbopac PA1 anion-exchange column wasemployed with an eluant consisting of 16% sodium hydroxide (200 mM) and84% deionized water. 3DF was detected electrochemically using a pulsedamperometric detector. Standard 3DF solutions spanning the anticipated3DF concentrations were run both before and after each unknown sample.

B. Measurement of Urine Creatinine

Urine creatinine concentrations were determined by the end-pointcolormetric method (Sigma Diagnostic kit 555-A) modified for use with aplate reader. Creatinine concentrations were assessed to normalize urinevolumes for measuring metabolite levels present therein.

C. Measurement of Albumin in the Urine

To assess albumin levels in the urine of the test subjects, spot urineswere collected and immunoephelometry performed on a BN 100 apparatuswith the N-albumin kit (Behring). Anti-albumin antibodies arecommercially available. Albumin levels in urine may be assessed by anysuitable assay including but not limited to ELISA assays,radioimmunoassays, Western and dot blotting.

Based on the data obtained in the study of the Joslin Diabetes Centerpatients, it appears that elevated levels of urinary 3DF are associatedwith progression to microalbuminuria in diabetes. This observationprovides a new diagnostic parameter for assessing the likelihood ofprogression to serious kidney complications in patients afflicted withdiabetes.

EXAMPLE 15 3-O-Methyl Sorbitollysine Lowers Systemic Levels of 3DG inNormal and Diabetic Rats

A cohort of twelve diabetic rats was divided into two groups of six. Thefirst group received saline-only injections, and the second receivedinjections of 3-O-methyl sorbitollysine in saline solution. The sameprocedure was conducted on a cohort of twelve non-diabetic rats. Assummarized in Table C, within one week, the 3-O-methyl sorbitollysinetreatment significantly reduced the plasma 3DG levels as compared to therespective saline controls in both diabetic and non-diabetic rats. TABLEC 3-O-Methyl sorbitollysine reduces plasma 3DG levels in diabetic andnon-diabetic rats. Diabetic Rats Non-diabetic Rats Plasma, Day 8 Plasma,Day 8 Control (n = 6) 0.94 ± 0.28 μM 0.23 ± 0.07 μM 3-O-methylSorbitollysine 0.44 ± 0.10 μM 0.13 ± 0.02 μM (n = 6) Percent reduction53% 43% t-test P = 0.0006 P = 0.0024

The ability of 3-O-methyl sorbitollysine to reduce systemic 3DG levelssuggests that diabetic complications other than nephropathy (e.g.,retinopathy and stiffening of the aorta) may also be controllable byAmadorase inhibitor therapy.

EXAMPLE 16 Locus of 3-O-Methyl Sorbitollysine Uptake In Vivo is theKidney

Six rats were injected intraperitoneally with 13.5 mmoles (4.4 mg) of3-O-methyl sorbitollysine. The rats' urine was collected for 3 hours,after which the rats were sacrificed. The tissues to be analyzed wereremoved and freeze clamped in liquid nitrogen. Perchloric acid extractsof the tissues were used for metabolite analysis. The tissues examinedwere taken from the brain, heart, muscle, sciatic nerve, spleen,pancreas, liver and kidney. Plasma and urine were also analyzed.

The only tissue extract found to contain 3-O-methyl sorbitollysine wasthat of the kidney. The urine also contained 3-O-methyl sorbitollysine,but plasma did not. The percentage of the injected dose recovered fromurine and kidney varied between 39 and 96%, as shown in Table D, below.TABLE D nmols nmols nmols total % 3OMeSL* 3OMeSL 3OMeSL 3OMeSL 3OMeSLRat # Injected in urine in kidneys recovered recovered 2084 13500 294010071 13011 96.4 2085 13500 1675 6582 8257 61.2 2086 13500 1778 53737151 53.0 2087 13500 2360 4833 7193 53.3 2088 13500 4200 8155 12355 91.52089 13500 1355 3880 5235 38.8*3-O-methyl sorbitollysine

While certain embodiments of the present invention have been describedand/or exemplified above, various other embodiments will be apparent tothose skilled in the art from the foregoing disclosure. The presentinvention is, therefore, not limited to the particular embodimentsdescribed and/or exemplified, but is capable of consideration variationand modification without departure from the scope of the appendedclaims.

1. A method for alleviating the deleterious effects of 3-deoxyglucosone(3DG) in a patient, said 3DG being produced by the enzymatic conversionof fructose-lysine to fructose-lysine-3-phosphate, said methodcomprising administering to said patient an inhibitor of said enzymaticconversion of fructose-lysine to fructose-lysine-3-phosphate saidinhibitor being a compound having the formula:

wherein X is a divalent moiety selected from the group consisting —NR′—or —O—, R′ being selected from the group consisting of H, and linear orbranched chain alkyl group (C₁-C₄) and an unsubstituted or substitutedaryl group (C₆-C₁₀) or aralkyl group (C₁-C₁₀); R is a substituentselected from the group consisting of H, an amino acid residue saidamino acid including said NR′ moiety, a polyamino acid residue saidpolyamino acid including said NR′ moiety, a peptide chain, a linear orbranched chain aliphatic group (C₁-C₈), which is unsubstituted orsubstituted with at least one nitrogen or oxygen-containing substituent,a linear or branched chain aliphatic group (C₁-C₈), which isunsubstituted or substituted with at least one nitrogen- oroxygen-containing substituent and interrupted by at least one —O—, —NH—,or —NR″— moiety, R″ being linear or branched chain alkyl(C₁-C₆) and anunsubstituted or substituted aryl group C₆-C₁₀) or aralkyl group(C₁-C₁₀), with the proviso that when X represents —NR′—, R and R′,together with the nitrogen atom to which they are attached, may alsorepresent a substituted or unsubstituted heterocyclic ring having from 5to 7 ring atoms, with at least one of nitrogen and oxygen being the onlyheteroatoms in said ring, said aryl group (C₆-C₁₀) or aralkyl group(C₇-C₁₀) and said heterocyclic ring substituents being selected from thegroup consisting of H, alkyl(C₁-C₆), halogen, CF₃, CN, and—O-alkyl(C₁-C₆); R₁ is a polyol moiety having 1 to 4 linear carbonatoms, Y is a hydroxymethylene moiety

Z is selected from the group consisting of —H, —O-alkyl (C₁-C₆),-halogen —CF₃, —CN, —COOH and —SO₃H₂ and the sterooisomers andpharmaceutically acceptable salts of said compound.
 2. The method ofclaim 1, wherein said inhibitor is a polyolysine or meglumine.
 3. Themethod of claim 1, wherein said polyolysine is selected from the groupconsisting of sorbitollysine, mannitollysine, galactitollysine and3-O-methyl sorbitollysine.
 4. The method of claim 1, wherein saidinhibitor is 3-O-methylsorbitollysine.
 5. The method of claim 1, whereinsaid deleterious effects of 3DG comprise pathological conditionsattributable to the formation of AGE proteins.
 6. The method of claim 5,wherein said pathological condition is selected from the groupconsisting of hypertension, stroke, neurodegenerative disorder,circulatory disease, glycogen storage disease, atherosclerosis,osteoarthritis and cataracts.
 7. The method of claim 6, wherein saidinhibitor is administered for alleviation of a neurodegenerativedisorder, said disorder being senile dementia of the Alzheimers type. 8.The method according to claim 1, wherein said inhibitor is orallyadministered.
 9. A method for alleviating the deleterious effects of3-deoxyglucosone (3DG) in a patient, said 3DG being produced by theenzymatic conversion of fructose-lysine to fructose-lysine-3-phosphate,said method comprising administering to said patient, an inhibitor ofsaid enzymatic conversion of fructose-lysine tofructose-lysine-3-phosphate.
 10. A method according to claim 9, whereinsaid inhibitor is of formula:

wherein X is a divalent moiety selected from the group consisting —NR′—or —O—, R′ being selected from the group consisting of H, and linear orbranched chain alkyl group (C₁-C₄) and an unsubstituted or substitutedaryl group (C₆-C₁₀) or aralkyl group (C₇-C₁₀); R is a substituentselected from the group consisting of H, an amino acid residue saidamino acid including said NR′ moiety, a polyamino acid residue saidpolyamino acid including said NR′ moiety, a peptide chain, a linear orbranched chain aliphatic group (C₁-C₈), which is unsubstituted orsubstituted with at least one nitrogen or oxygen-containing substituent,a linear or branched chain aliphatic group (C₁-C₈), which isunsubstituted or substituted with at least one nitrogen- oroxygen-containing substituent and interrupted by at least one —O—, —NH—,or —NR″— moiety, R″ being linear or branched chain alkyl(C₁-C₆) and anunsubstituted or substituted aryl group C₆-C₁₀) or aralkyl group(C₇-C₁₀), with the proviso that when X represents —NR′—, R and R′,together with the nitrogen atom to which they are attached, may alsorepresent a substituted or unsubstituted heterocyclic ring having from 5to 7 ring atoms, with at least one of nitrogen and oxygen being the onlyheteroatoms in said ring, said aryl group (C₆-C₁₀) or aralkyl group(C₇-C₁₀) and said heterocyclic ring substituents being selected from thegroup consisting of H, alkyl(C₁-C₆), halogen, CF₃, CN, and—O-alkyl(C₁-C₆); R₁ is a polyol moiety having 1 to 4 linear carbonatoms, Y is a hydroxymethylene moiety

Z is selected from the group consisting of —H, —O-alkyl (C₁-C₆),-halogen —CF₃, —CN, —COOH and —SO₃H₂ and the sterooisomers andpharmaceutically acceptable salts of said compound.
 11. The method ofclaim 9, wherein said inhibitor is a polyolysine or meglumine.
 12. Themethod of claim 9, wherein said inhibitor is 3-O-methylsorbitollysine.13. The method of claim 9, wherein said deleterious effects of 3DGcomprise pathological conditions attributable to the formation of AGEproteins.
 14. The method of claim 13, wherein said pathologicalcondition is selected from the group consisting of hypertension, stroke,neurodegenerative disorder, circulatory disease, glycogen storagedisease, atherosclerosis, osteoarthritis and cataracts.
 15. The methodof claim 14, wherein said inhibitor is administered for alleviation of aneurodegenerative disorder, said disorder being senile dementia of theAlzheimers type.
 16. The method according to claim 9, wherein saidinhibitor is orally administered.
 17. The method according to claim 1,wherein the inhibitor is meglumine.
 18. The method according to claim 9,wherein the inhibitor is meglumine.